GSM7977018: Cornea, AALlow; Homo sapiens; RNA-Seq
Cells were harvested directly from donor corneas as above and suspended in flow buffer (2% FBS in DPBS no calcium, no magnesium supplemented with 1mM EDTA). Cells were incubated with 1µg/mL fluorescein conjugated AAL on ice for 30 minutes, with gentle mixing every 10 minutes. Cells were collected by centrifugation, washed, centrifuged, and resuspended in flow buffer. Immediately before sorting, cells were again filtered with a cell strainer.  Addition of 3 µM propidium iodide (Thermo Fisher Scientific) was used to exclude dead cells. The stained cells were sorted by a Coulter ELITE cell sorter (Miami, FL, USA) calibrated for two-color fluorescence. The cells were sorted into two populations, cells with relatively low AAL binding (AALlow)and cells with relatively high AAL binding (AALhigh). Single cell suspensions of AAL-sorted corneal limbal epithelial cells were passed through a 35 µm nylon cell strainer, centrifuged for 5 min at 400× g and resuspended in 1 mL PBS on ice. The cells were again centrifuged for 5 min at 400× g and resuspended in ice-cold PBS at a ratio of 200 μL PBS per 1 × 106 cells followed by the dropwise addition of ice-cold methanol, at a ratio of 800 μL per 1 × 106 cells. The cells were stored at − 80 °C until sequencing. Single-cell mRNA sequencing was performed by the BWH Center for Cellular Profiling according to standard 10X Genomics 3' V3.1 chemistry protocol. Prior to loading the cells on the 10X Chromium controller, cells were rehydrated in rehydration buffer. Cells were then counted to assess cell integrity and concentration. Approximately 3,400 AALlow and 1,200 AALhigh cells from three donor corneas were separately loaded and the resulting sequencing libraries were prepared following a standard 10X Genomics protocol. Briefly, samples were processed through GEM wells and libraries generated and sequenced.